Automated High-throughput Behavioral Analyses in Zebrafish Larvae

The overall goal of this procedure is to identify behavioral defects in zebrafish larvae after treatment with toxicants or pharmaceuticals. This is accomplished by first collecting and treating the embryos or larvae with chemicals up to the first seven days of development. The second step is to make agro swimming lanes using specially designed molds.

Next, the treated larvae are placed in the agros lanes for behavioral analysis using time-lapse photography. The final step is to import the images into image J and use a specific macro written in-house in order to gather XY coordinate data of the larvae in the lanes. Ultimately, the high throughput behavioral assay is used to find differences in avoidance and figma axis behavior, as well as in swim speed and rest between the treatment groups.

The main advantage of this technique over existing methods like those commercially available is that we can use our method to observe more complex behaviors. It is also inexpensive to build and easy to set up. This method can help answer key questions in the field of behavioral neuroscience, such as how toxicants affect brain development and behavior during early embryonic exposure.

It can also provide insight into subtle brain defects in humans, which can not be found by other current toxicity screens. Generally, individuals new to this method will struggle because catching the larvae can be difficult. Visual demonstration of this method is critical as the steps have a learning curve since the protocol involves custom built systems.

To begin this procedure, insert glass Pyrex dishes with fake grass made from green yarn into the tanks. At dawn, leave the dishes in the tanks for two hours in order to collect zebrafish embryos. Pour the glass dishes containing the embryos over a handheld strainer rin with deionized water.

Next, grow the embryos in egg water containing 60 milligrams per liter of instant ocean in deionized water and 0.25 milligrams per liter of methylene blue, which is used as a mold inhibitor. Embryos can be housed at 50 to 60 larvae per 50 milliliters or in larger breeding tanks until embryos are ready to be treated with toxicants. Depending upon the hypothesis of the individual experiment, embryos can be treated immediately or during specific stages of development using toxicants or pharmaceuticals, which are usually dissolved in DMSO and then further diluted directly in the egg water medium.

The blue color is used for video demonstration purposes during embryonic and larva treatment. The larvae and embryos can be housed in deep Petri dishes at a density of about 50 to 60 larvae per 50 milliliters until the behavioral analysis at seven days post fertilization change the egg water solution at least every other day to avoid fungal or bacterial growth from the dead embryos. Plastic molds were fabricated to create lanes using aros, which is poured into single well plastic plates from Thermos Scientific.

The molds contain five lanes in which the sides are angled at 60 degrees. See the text protocol accompanying this video for the dimensions of the molds and the plates? First, prepare the aros by melting 0.8%aros in deionized water with 60 milligrams per liter of instant ocean.

Next, generate the lanes by pouring 50 milliliters of the melted aros into a single well plate. Then very slowly place the mold on top of the liquid aros who eliminate any bubble formation. Remove the mold when the aros has cooled.

The aros lanes can be stored at room temperature with the lids on the dishes for up to 36 hours. For high throughput behavioral analysis, imaging cabinets are prepared with a 15 megapixel digital camera attached to the top of the cabinet facing downward at the bottom of the cabinet. A 15.6 inch screen laptop is placed with the screen facing up.

In order to avoid overheating the larvae, it is best to use a laptop with a screen temperature that does not go above 28 degrees Celsius. Place a plastic diffuser on top of the laptop screen on which the agros plates will be placed. The diffuser will prevent more patterns in the images collected.

To begin image capture, carefully move the larvae from the Petri dish to the agros lane to help reduce larvae stress. Up to 20 larvae can be placed in each lane, but five larvae per lane are typically used to facilitate the most accurate tracking of swim speed and to reduce the number of larvae that are needed per experiment, fill the lanes with egg water, with or without pharmaceuticals or toxicants depending upon the experiment. Do not fill up the lanes all the way until they are placed in the imaging cabinets to prevent overflow.

Allow 10 minutes for the larvae to acclimate after the acclimatization period. Position four plates by hand directly on top of the laptop screen. At this time, the lanes can be topped off with egg, water or chemical treatment so that it is level with the top of the lane to eliminate shadows on the edges of the lanes.

In the images program the computer for time-lapse photography, taking pictures every six seconds for a total of 300 images per experiment. Set the camera at a lower resolution for imaging at video speed. While the lower resolution limits the recordings to a single multi-well plate.

The video recordings are appropriate for imaging rapid swimming events. Use a PowerPoint presentation as an aversive stimulus for the larvae in this demonstration. The PowerPoint starts with a blank white background for 15 minutes, followed by 15 of a moving red bar on the top half of the plate.

To avoid evaporation of the liquid within the aros lanes, keep the maximal imaging time to below one hour. Open the images with image J in this demonstration. An in-house macro for five lane larval analysis is used and can be made available to any user.

Use the prompts in the macro to set the parameters, such as number of images and color to be subtracted. The macro then automatically splits the color channel so that the red color can be removed. Subtracts the background applies a threshold and identifies the larvae by particle analysis.

After all of the images are run through the image J Macro, a results file will be displayed and will contain the XY coordinates of the individual larvae for each image, along with the image number and the lane number. Save the results file in an Excel format and sort based upon blank background versus the moving bar background, and then the well number. Use an Excel template that has equations built in to automatically determine placement of larvae in the wells distance between larvae, speed of movement, and amount of rest.

The most current Excel template created in the Cretin lab is available upon request build graphs showing various treatment groups into the Excel sheet, along with T-test for comparison between treatment groups and controls. Further statistical analysis can be performed using SPSS statistics program. Results are shown for larvae treated with egg water and DMSO as controls and varying concentrations of an organophosphate pesticide commonly found in non-organic foods.

Here the white bars show data from larvae exposed to a blank background, and the red bars show data from the larvae exposed to the red moving bar.Control. Larvae grown in egg water show an increased preference to be down in the dish after they are presented with the moving red bar. Similar results are obtained when larvae are grown in egg water containing one microgram per milliliter of DMSO, A solvent that is commonly used to dissolve various pharmaceuticals and toxicants as 1000 x stock solutions.

The graphs indicate the measurements that can be obtained from behavioral analysis, including percentage of larvae down in the lane, percentage of larvae on the end of the lane, percentage of larvae on the edge of the lane, distance between fish swim speed of the larvae, and percentage of time the larvae are at rest. The results shown are a sampling from one experiment. However, when repeated, the results indicate that swim speed and figma behavior is altered by low concentrations of organophosphate pesticides, which mimic levels in human food Once mastered, the imaging and imaging analysis can be done in an hour and a half if it is performed properly.

The most important aspect of this procedure is to remember to keep the cultural medium clean by replacing with new medium daily.